MECHANISM OF AMMONIA TOLERANCE IN THE GULF …：在海湾氨耐受机制….doc

MECHANISM OF AMMONIA TOLERANCE IN THE GULF TOADFISHVeauvy C.M. Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, 4600 Rickenbacker causeway, Miami Florida 33149, USA. phone: 305 361 4859 fax: 305 361 4600 email: McDonald M.D., Walsh P.J., Van Audekerke J., Vanhoutte G., Van Camp N., Van der Linden A. EXTENDED ABSTRACT ONLY DO NOT CITEIntroductionThe gulf toadfish, Opsanus beta, is more tolerant to high ammonia concentration in the blood than most fish or mammals. The 96hLC50 for most fish expressed as un-ionized ammonia is below 200 mol/L whereas for gulf toadfish it is 600 mol/L (equivalent to 10 mM total ammonia) (Wang and Walsh, 2000). In patients, cerebral edema (one of the symptoms of hyperammonemia) is observed with concentrations of total ammonia in the plasma being 600 mol/L. Hyperammonemic events in mammals can induce brain swelling (Brusilow, 2002). It is believed that an enzyme, glutamine synthetase, located in the astrocyte cells of the brain converts excess ammonia into glutamine. It is hypothesized that water enters brain tissue to relieve the osmotic imbalance created by the accumulation of glutamine leading to brain swelling. Studies in rodents using the drug methionine sulfoximine, an irreversible blocker of the enzyme glutamine synthetase, partly prevent the accumulation of glutamine and alleviate brain swelling during hyperammonemia (Takahashi et al., 1991). AimTo understand the mechanism by which Opsanus beta can tolerate hyperammonemia in the context of the brain glutamine hypothesis, we used magnetic resonance imaging technique (MRI) to monitor changes in water content in the brain. The MRI technique gives information about the movement of water between the intracellular and extracellular milieu of cells (apparent diffusion coefficient) and about the movement of water in and out of the whole brain tissue (T2 parameter) (Van der Linden et al., 2001).MethodsWe exposed toadfish to two types of ammonia treatments in seawater: a chronic exposure to a sublethal concentration of ammonium chloride 3.5 mM (1/3 the 96hLC50 value) for 16 and 40 hours and an acute exposure to 10, 20, and 30 mM ammonium chloride consecutively for one hour in each concentration. The values obtained for the apparent diffusion coefficient and the T2 parameter in the hyperammonemic state were compared to values obtained in control situations. For the chronic exposure, plasma and tissue samples (brain, liver, gills and muscle) were collected at 16 and 40 hours to monitor the levels of glutamine, glutamate, ammonia and urea in the fish.ResultsThe results from the MRI analysis show no significant changes in the apparent diffusion coefficient for the chronic and acute exposure suggesting that water molecules didnt shift from the extracellular to the intracellular compartment of the cell. However, a significant decrease in T2 parameter for the chronic exposure suggests a loss of water to the whole brain. This result was corroborated by a significant increase in plasma osmolality for fish exposed for 16 and 40 hours suggesting whole body dehydration. Measurements of blood pH and plasma bicarbonate concentration show no disturbance in the acid-base status of the fish. There was no significant change in the T2 parameter during the acute exposure.ConclusionToadfish and rodents seem to have a different response to hyperammonemia in the context of brain water. While hyperammonemic rodents tend to gain water to the brain leading to brain edema, toadfish lose water from the brain and display whole body dehydration without an impairment of the acid-base regulatory mechanism located at the gill surface. Whole brain glutamine concentration in the toadfish is significantly raised during high ammonia exposure (Wang and Walsh, 2000). However, this increase is not as pronounced as for rats. Comparing two studies (Brusilow, 2002 and Wang and Walsh, 2000) where rats and toadfish were made hyperammonemic by raising the concentration of ammonia in the plasma to about 12 times its original concentration, rat brain glutamine concentration raised by 3.5 times its control value while toadfish brain glutamine concentration raised by only 1.2 times its control value. It is possible that toadfish could survive the toxic effect of ammonia to the brain by preventing brain glutamine to accumulate to a certain threshold value.AcknowledgementsThe research was supported by the National Institute of Health grant to PJW ES-11005 and the National Institute of Environmental and Health Sciences to PJW ES 05705.ReferencesBrusilow S.W., 2002. Hyperammonemic encephalopathy. Medicine 81(3): 240-249.Takahashi H., Koehler R.C., Brusilow S.W., Traystman R.J., 1991. Inhibition of brain glutamine accumulation prevents cerebral edema in hyperammonemic rats. American Journal of Physiology 261: H825-829.Van der Linden A., Verhoye M., and Nilsson G.E., 2001. Does anoxia induce cell swelling in c